Spider toxins and methods for their use as blockers of amino acid receptor function

ABSTRACT

Methods and compositions for blocking various channels and receptors within an organism are provided. For example, a toxin was isolated from the Argiope aurantia spider. This toxin was found to have a reversible effect on excitatory amino acid receptors. Another toxin having a molecular weight of from approximately 5,000 to approximately 7,000 daltons was isolated from the Hololena curta spider. This toxin was found to have an irreversible effect on excitatory amino acid receptors. 
     The present invention further relates to methods of treating heart and neurological diseases by applying the toxins isolated and identified. In particular, the low molecular weight toxin from Agelenopsis aperta may provide a treatment of certain heart conditions such as arrhythmia, angina, hypertension, and congestive heart failure. In addition, the toxins may provide beneficial effects on certain neuological conditions including seizures. It is also found that the toxins having an irreversible effect are effective as tags in probing the various components within the calcium channels and excitatory amino acid receptors and are effective insecticides and anthelmintics.

This application is a continuation of application Ser. No. 06/921,218,filed Oct. 20, 1986, now U.S. Pat. No. 4,925,664.

BACKGROUND

1. The Field of the Invention

The present invention generally relates to the isolation of certaintoxins from spider venoms and the use of those toxins as inhibitors ofthe functions of ion channels and neurotransmitter receptors. Inparticular, the present invention relates to spider venom toxins andtheir use as blockers of calcium channels and excitatory amino acidreceptors in the cardiovascular, central nervous, and neuromuscularsystems of organisms, including humans.

2. The Background of the Invention

Movement of calcium ions across cell membranes is a critically importantevent in the normal functioning of excitable tissues such as vascularsmooth muscle, cardiac muscle, and the central nervous system. Influx ofcalcium ions through specialized channels in the cell membranesregulates release of substances such as hormones and neurotransmitters.

The movement of calcium ions also regulates contraction of heart muscleand of vascular smooth muscle in the wall of blood vessels. Abnormalinflux of calcium ions has been reported to play a role in thepathogenesis of various cardiovascular disorders (e.g., anoxic/ischemicheart disease), and drugs capable of blocking the movement of calciumthrough calcium channels have been used for treatment of cardiacarrhythmias, coronary artery disease, and cardiomyopathy.

The currently used drugs, however, have non-specific physiologicaleffects and varying tissue specificities that can lead to undesirableside-effects in patients. Moreover, there are several known subtypes ofcalcium channel with varying physiological action and no drug thatspecifically blocks certain of these subtypes is known.

In the nervous system, calcium influx into the presynaptic nerveterminal via calcium channels is a necessary prerequisite for therelease of chemical neurotransmitter at synapses and thus for the properfunctioning of these synapses. Lowering of the extracellular calciumconcentration is routinely used by neurophysiologists to reduce orabolish synaptic transmission in isolated pieces of nervous tissue.

It has not been possible, however, to specifically affect synaptictransmission in vivo in the central nervous system ("CNS") bymanipulating the function of neuronal calcium channels. With theexception of the omega-conotoxin recently isolated from the venom of themarine snail Conus geographus, no drug with sufficiently specific orpotent effects on CNS calcium channels is known.

Abnormal influx of calcium is thought to be very important in thepathogenesis of several CNS disorders, including anoxic/ischemic(stroke) damage, epilepsy, and the neuronal death associated withchronic epilepsy. Again, the paucity of chemical agents that potentlyand specifically block CNS calcium channels has prevented thedevelopment of an effective drug therapy for these prevalentneurological problems.

In addition, excitatory amino acids ("EAA"), most notably glutamate andaspartate, are the predominant excitatory neurotransmitter in thevertebrate (including human) CNS. As such, they play a fundamental rolein the many functions of the normal nervous system. EAA's are releasedfrom presynaptic nerve terminals and, after diffusing across thesynaptic cleft, contact special EAA receptor molecules in thepostsynaptic cell membrane. These receptors indirectly influence theflow of various ions across the cell membrane and thus contribute toproduction of an electrical response to the chemical message deliveredby EAA neurotransmitter molecules. A number of common and very seriousneurological problems involve abnormal function of EAA synapses. Theseinclude epilepsy, several degenerative disorders such as Huntington'sdisease, and neuronal death following stroke.

Unfortunately, there are very few chemical agents which are potent andselective blockers (that is "antagonists") of EAA receptors. This hasseverely hampered research on the normal function of EAA's and limitedtherapeutic approaches to disorders involving EAA's.

One notable limitation of the currently available EAA receptorantagonists is the lack of any drugs with very high affinity for thereceptor. A drug with high affinity for the receptor could be expectedto produce irreversible blockade of synaptic transmission. When labeledwith some tracer molecule, such a drug would provide a reliable way oftagging receptors to permit measurement of their number and distributionwithin cells and tissues. These features would have very valuableconsequences for research on EAA neurotransmission and for thedevelopment of therapeutic agents to treat EAA dysfunction in humans andanimals.

Arthropod animals, including insects, and certain parasitic worms useEAA's as a major chemical neurotransmitter at their neuromuscularjunction and in their CNS. Because of the damage done by insect pestsand the prevalence of parasitic worm infections in animals and humans inmany countries, there is a constant need for potent and specific newpesticides and anthelmintic drugs that are non-toxic to humans, pets,and farm animals.

As described above, it would be a very considerable improvement in theart if it were possible to develop chemical agents that specifically andpotently block calcium channel function in the CNS, cardiac muscle, andvascular smooth muscle. In particular, it would be an advancement in theart to provide a specific blocker for specific subtypes of calciumchannel. Similarly, it would be an advancement in the art to provide aspecific blocker of calcium channels in the CNS.

It would be a further significant advancement in the art if chemicalcompositions could be found that specifically bind to and block thefunction of EAA in the CNS of vertebrates (including humans) and in thenervous systems of invertebrates. It would be a further advancement inthe art to provide calcium channel blockers and EAA receptor blockerswhose actions were selectively reversible or irreversible for use inclinical settings or as research tools. It would be an additionaladvancement in the art to provide calcium channel blockers and EAAreceptor blockers that were acceptable for use as insecticides andanthelmintics.

Such chemical compositions and methods for their use are disclosed andclaimed below.

BRIEF SUMMARY AND OBJECTS OF THE INVENTION

The present invention is related to the isolation, identification, anduse of various spider venoms and toxins contained within these venoms.In particular, the present invention is related to the isolation and useas calcium channel blockers or excitatory amino acid receptor blockersof certain toxins from spider venom.

As discussed above, calcium channels are intimately involved in thefunctions of the cardiovascular system since calcium influx affectscontraction of cardiac muscle and vascular smooth muscle. Similarly,calcium influx into nerve cells is required for the release of chemicalneurotransmitter substances at synapses and, therefore, for the normalfunctioning of the nervous system. Abnormal calcium influx into cells isassociated with serious cardiovascular and neurological disorders.

As also discussed above, EAA receptors mediate synaptic transmission atthe most common excitatory chemical synapse in vertebrate brain and,therefore, play a profound role in normal functioning of the nervoussystem. Dysfunction of EAA neurotransmission is associated with avariety of serious neurological disorders. The present invention isrelated to obtaining toxins from spider venoms, which toxins havespecific and potent blocking effects on calcium channels or on EAAreceptor within the organism.

Within the scope of the present invention, spider venom is obtained bymilking spiders of various species. That is, the spider venom isobtained by electrical stimulation of the spider to cause release of thevenom and subsequent suction in order to collect the released venom.This assures that impurities, which have traditionally been containedwithin spider venoms obtained by conventional techniques, areeliminated.

Spider venoms are known to be a complex mixture of enzymes, peptidetoxins, nucleotides, free amino acids, and other molecules. As a result,in order to obtain useful spider toxins it is necessary to separate thevarious components of the whole spider venom. According to oneembodiment of the present invention, whole venoms are fractionated bygel filtration to separate components of the venom by relative molecularmass. It will be appreciated, however, that any type of fractionationtechnique or other technique may be useful to obtain the spider venomtoxins necessary for use in the present invention.

A group of specific spider venoms have been isolated and usedextensively in the context of the present invention. The spiders thathave been used within the scope of the present invention includeAgelenopsis aperta, Argiope aurantia, and Hololena curta. Agelenopsisand Hololena are members of the funnel-web grass spider familyAgelenidae and are commonly found in meadows and other grassy areaswithin the western United States.

Four specific toxins that fall within the scope of the present inventionhave been isolated from the Agelenopsis, Argiope, and Hololena spiders.In particular, a first high molecular weight toxin has been isolatedfrom the Agelenopsis aperta spider. For ease of identification thistoxin will be sometimes generally referred to as "AG1" during thepresent description of the invention.

It has been found that AG1 produces "irreversible" blockade of synaptictransmission under certain conditions without affecting axonalconduction of action potentials. The AG1 toxin affects transmission ofthe nerve impulse across the synapse. For the purpose of the presentdiscussion, irreversible blockade is defined as blockade that is notreversed during the useful life of the various cell and tissuepreparations used in experimentation with the various toxins.

AG1 is also found to irreversibly block transmission in certain centralnervous system cells by blocking the transient calcium current. It isparticularly noteworthy that AG1 is not acutely toxic to the cellstested and does not affect the electric excitability of the neuronsthemselves. Thus, suggests that AG1's effects are not produced by acutecytotoxic action. Simply stated, CNS transmission is blocked withoutdamaging the cells involved.

A second toxin which falls within the scope of the present invention isa low molecular weight toxin also isolated from the Agelenopsis apertaspider, designated AG2 for purposes of this discussion. This toxin has amolecular weight of between 200 and 1,000 daltons. In particular, it ispresently believed that the toxin has a molecular weight ofapproximately 600 daltons.

AG2 has been found to "reversibly" block synaptic transmission and itseffects are diminished by increasing calcium concentrations. Inexperiments on cardiac muscle this toxin has also been shown to blockthe slow inward calcium current. Furthermore, small quantities of thistoxin injected intravenously into rats significantly reduce the severityand duration of seizures induced by intravenously injected kainic acid.As a result, it can be seen that this toxin affects both the CNS and thecardiovascular system.

A further toxin within the scope of the present invention is a lowmolecular weight toxin isolated from the Argiope aurantia spider. Forthe purpose of this discussion this toxin is sometimes referred to asARI. ARI has been found to be an EAA receptor blocker. This toxinproduces reversible blockade of transmission of central nervous systemcells.

A fourth toxin within the scope of the present invention has beenisolated from the Hololena curta spider and has a molecular weightbetween 5,000 and 7,000 daltons. This toxin is sometimes designated HOIfor the purpose of this discussion. HOI has been found to irreversiblyand potently block CNS excitatory amino acid receptor channels withoutacute cytotoxicity. It will be appreciated that such a toxin isparticularly adaptable for use in a research setting. Since the toxin'seffect is irreversible it can be used as a tag to identify particularchemical species within cells, tissues, or organs.

The toxins discussed herein are potent and effective calcium channel orEAA receptor blockers when compared to conventional blockers. A varietyof excitatory amino acid agonists and antagonists have been tested inthe art for their ability to suppress synaptic transmission in the chickcochlear nucleus.

The most potent conventional antagonists, which are chemical analogs ofacidic amino acids, require concentrations in the millimolar range tocompletely suppress the transmission. Even then, the effect of thesedrugs is complete reversed within 20 minutes. In contrast, partiallypurified spider toxins within the scope of the present invention canirreversibly block transmission with concentrations conservativelyestimated to be less than one micromolar.

As a result, it will be appreciated that the present invention providesthe capability to block, either reversibly or irreversibly, specificcalcium channels or EAA receptors. For example, calcium channels withinthe cardiovascular system may be blocked with specificity. Likewise,central nervous system calcium channels may also be blocked withspecificity. In the alternative, when applying AG1 toxin, both centralnervous system and cardiac systems may be blocked using the same toxin.

It will also be appreciated that the present invention provides theability to effectively block specific channels using a very smallconcentration of toxin. The effectiveness of the present invention is atleast one order of magnitude higher than the most potent currently knownblocker of EAA transmission.

As discussed above, many uses for specific channel blockers are knownand many other uses are possible. For example, a channel blocker whichimpacts the cardiovascular system may be an effective treatment for highblood pressure. Such a channel blocker may also have usefulness intreating arrhythmia, angina, and other types of heart disease.

As a further matter, such channel blockers may be effective treatmentsof post cardiac arrest cell damage. It is known in the art that celldamage to the heart following a heart attack is to a significant degreedue to increases in intracellular calcium concentration. As a result, acalcium channel blocker may help prevent cell damage that wouldotherwise occur.

Similarly, specific channel blockers with activity on the centralnervous system may have the potential to treat various neurologicaldiseases. It has been found, for example, that these channel blockersmay act as a treatment of epilepsy. In addition, channel blockers of thetype disclosed in the present invention may also be used in treatmentsof degenerative central nervous system diseases such as Alzheimer'sdisease.

As a related matter, it will be appreciated that EAA receptor blockersand calcium blockers of the type disclosed herein may be used asinsecticides and anthelmintics because the molecules disclosed withinthe present invention can be applied to insects with great effectivenessfor eliminating those insects and related pests.

As a result, it is a primary object of the present invention to provideEAA receptor blockers and calcium channel blockers and methods for theiruse which have specific and identifiable effects on an organism.

It is a further object of the present invention to provide specificcalcium receptor blockers.

Similarly, it is an object of the present invention to provide specificexcitatory amino acid receptor blockers.

Another object of the present invention is to provide a specific calciumchannel blocker which affects only the central nervous system.

Additionally, it is an object of the present invention to providecalcium channel blockers which have effects on the cardiovascularsystem.

It is another object of the present invention to provide calcium channelblockers and EAA receptor blockers which are selectively reversible orirreversible in their effects for use as research tools and for use inthe clinical setting.

Finally, it is an object of the present invention to provide calciumchannel blockers and EAA receptor blockers which are usable asinsecticides and anthelmintics.

These and other objects of the present invention will become apparentupon reading the following detailed description and appended claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As discussed above, the present invention is related to new and uniquecalcium channel blockers and EAA receptor blockers, methods for theirisolation, and methods of application of such molecules. In particular,the present invention relates to the use of isolated toxins obtainedfrom spider venom for use as specific calcium channel or EAA receptorblockers.

It has been found within the scope of the present invention that certainspider venoms may selectively act on the central nervous system.Likewise, certain spider toxins within the scope of the presentinvention selectively act on the cardiovascular system. Moreparticularly, it has been found that spider venoms can have specificactivities on calcium channels and/or excitatory amino acid receptorswithin the organism.

The toxins within the scope of the invention also eliminate many of theside effects encountered in prior art calcium channel blockers and EAAreceptor blockers. The toxins of the present invention are not expectedto significantly interact with other types of drugs because of theirspecific activity.

In addition, it is possible using the compositions of the presentinvention to choose either reversible or irreversible toxins dependingon the preferred area of use. It is expected that for most clinicalapplications toxins with a reversible activity will be preferred. At thesame time, toxins with irreversible activity may be preferred in theresearch setting where it is desirable to tag certain neurotransmitterreceptors or ion channels.

An additional benefit of the present invention is that the isolatedtoxins act without significant cytotoxicity. Thus, the toxins do notblock channels by destroying the cells within the systems in which theyare active. Additionally, the toxins of the present invention generallyact without affecting axonal conduction within the nervous system. Itwill be appreciated, therefore, that only the receptor or ion channel isaffected by the toxins which act on the central nervous system.

As mentioned above, toxins which have been identified and used withinthe scope of the present invention include those isolated from thespiders Agelenopsis aperta, Argiope aurantia, and Hololena curta. Asdiscussed briefly above, Agelenopsis and Hololena are members of thefunnel-web grass spider family Agelenidae which includes approximately1,000 species worldwide. These spiders are commonly found in meadows andother grassy areas in the western United States. These spiders arerather inconspicuous and develop flat funnel-shaped webs used to gatherprey. Thus, these spiders must move rapidly to seize, bite, andimmobilize their prey. Their venoms act rapidly and potently to paralyzeinsects.

Argiope is a member of the large family of orb-weaving spiders(Araneidae) which includes approximately 2,500 species. These aregenerally large and conspicuously decorated spiders that build large,symmetrical, and sticky webs capable of snaring flying insects.

I. TECHNIQUES FOR ISOLATION OF VENOMS

In order to avoid impurities within the spider venom and the isolatedtoxins, the spider venom which was used for the tests described belowwas electrically milked from the spiders using a method which employssafeguards to prevent contamination of the venom by abdominalregurgitate or hemolymph.

Once the spider venom is obtained by electrical milking techniques, itis further purified using gel filtration chromatography or other similarrelated techniques. In addition, it is frequently desirable for finalfractionation of the spider venom to be performed by high performanceliquid chromatography ("HPLC").

Thus, using the technique of electrically milking the spider coupledwith gel filtration chromatography and high performance liquidchromatograhy it is possible to obtain purified and usable spidertoxins. It will be appreciated, however, that other equivalenttechniques may also be employed within the scope of the presentinvention in order to isolate the spider toxins used.

II. SPECIFIC TOXINS WITHIN THE SCOPE OF THE INVENTION

While it will be appreciated that additional toxins may also fall withinthe scope of the present invention, the following is a sampling of someof the toxins which have been specifically identified and which have thecharacteristics required for a usable calcium channel blocker or EAAreceptor blocker as described above.

A. Agelenopsis aperta I (AG1)

Using the techniques described above relating to the collection ofvenom, a toxin has been isolated from the Agelenopsis aperta spiderhaving a molecular weight of between 5,000 and 10,000, and moreparticularly estimated to be approximately 6,000. It has been found thatAG1 blocks transmission in chick cochlear nucleus neurons in an in vitropreparation of chick brain stem. Blockade persists for the useful lifeof the preparation (8 to 12 hours) and is regarded as irreversible asthat term is defined herein. Transmission at the synapse used in theexperimental techniques is mediated by non-N-methyl-D-aspartate("non-NMDA") excitatory amino acid receptors.

It has been found that even extremely low concentrations of AG1 producecomplete blockade if applied for extended periods, suggesting that thetoxin binds very tightly and essentially nonreversibly on or within thecell In experiments performed using the toxin, it has been found thatthe toxin is very potent in that exposure of the tissue to a 0.8micromolar solution for even two minutes results in a complete blockadeof synaptic transmission.

Complete blockade using AG1 on central nervous system cells occurs inthe absence of presynaptic action potentials and the time course ofaction is unaffected by the rate of presynaptic stimulation. Partialblocks have been achieved by brief exposure to dilute AG1 toxin, werestable for at least one hour and were unaffected by increases ordecreases in stimulation rate. Partial blockade by AG1, however, can belargely reversed by increasing the extracellular calcium concentration.Subsequent reduction in calcium concentration, however, causes the postsynaptic response to decline to its previous level of partial blockade.

In the absence of toxin the same increase in calcium has no effect onthe amplitude of responses. These results indicate that this toxin actspresynaptically to produce long lasting blockade of transmission. Theeffects are independent of stimulation frequency, suggesting that thetoxin does not act primarily on synthesis or reuptake of transmitter.Calcium antagonism of the effects makes it improbable that the toxincauses massive release of transmitter.

In summary, it is found that this relatively high molecular weight toxinis antagonized by increasing calcium concentrations and producesirreversible blockade of synaptic transmission in cochlear chick nucleuswithout affecting the afferent volley. It is also found to irreversiblyblock transmission in chick spinal cord by blocking the transientcalcium current. In addition, this toxin has been found not to beacutely toxic and does not affect the electrical excitability ofcochlear nucleus neurons themselves, indicating that its effects are notproduced by acute cytotoxic action.

B. Agelenopsis aperta II (AG2)

A second low molecular weight toxin has also been isolated and purifiedfrom Agelenopsis aperta toxin. This toxin, like AG1, has been purifiedby gel filtration, ion exchange, and HPLC. Following such purification,AG2 has been found to have a molecular weight of between 200 and 1,000,and most probably about 600 daltons.

It has been found that AG2 is an effective blocker which is active bothin the cardiovascular system and the nervous system. In particular, ithas been found that AG2 reversibly blocks synaptic transmission in chickcochlear nucleus, rat hippocampus, mouse diaphragm, and chick cardiacmuscle. In addition, it has been found that in certain experimentalsettings AG2 is antagonized by increasing calcium concentrations.

In experiments performed on cardiac muscle it has been found that AG2blocks the slow inward calcium current mentioned above. As a result, itwill be appreciated that AG2 may be an effective calcium channel blockerin cardiac and vascular smooth muscle.

Additional experiments have been performed using AG2 in order todetermine its neurological effects. In particular, rats wereintravenously injected with kainic acid in order to artificially produceseizures. It was found in experiments where AG2 was injected into ratstreated with kainic acid, that AG2 significantly reduced the severityand duration of the seizures induced. AG2 also has a significant effecton the nervous system and is an attractive potential treatment forepilepsy, degenerative diseases such as Alzheimer's disease,anoxic/ischemic brain damage (stroke) and other neurological problems.

C. Argiope aurantia I (ARI)

It has been previously shown that transmission between the aviancochlear nerve and neurons of the cochlear nucleus is mediated bynon-N-methyl-D-aspartate receptors and, presumably, an excitatory aminoacid transmitter. The effect on transmission in those settings has beendetermined using Argiope aurantia toxin (ARI) obtained and purifiedusing the same general methods described above.

It has been found that when applying Argiope aurantia toxin to thecochlear nucleus that transmission is blocked within 15 minutes withoutaffecting the afferent volley. Recovery of transmission occurs after 45to 90 minutes of washout of the toxin.

Partial purification with gel filtration has shown that this particulartoxin is a low molecular weight fraction of between approximately 500and than 1,000 daltons, and likely about 660 daltons.

Experimentation using ARI toxin has indicated that there was no stimulusdependency in blockage of the avian cochlear nucleus by ARI. Increasingthe frequency of stimulation from 0.3 to 20 Hz had no effect on the rateof blockage and suppression is equally complete and rapid even in thetotal absence of presynaptic stimulation and spontaneous activity.

In summary, ARI is a relatively low molecular weight toxin. In tests runon the toxin, it has been found that the toxin produces a readilyreversible blockade of transmission in chick cochlear nucleus. As aresult, the toxin is expected to provide reversible blockade in variouscentral nervous system settings.

In addition, since ARI is an excitatory amino acid receptor blocker, itis expected to have devastating effects when applied to insects andother invertebrate animals if administered in sufficient quantities. Asa result, ARI would be an effective insecticide or anthelmintic.

D. Hololena curta I (HOI)

An additional active toxin has been isolated from the Hololena curtaspider. It is found that this toxin has a molecular weight of from about5,000 to about 7,000 daltons. This particular toxin has been found toblock postsynaptic response in chick cochlear nucleus. Furthermore, theeffectiveness of HOI toxin has been found to be irreversible over thelife of the tissue preparation but without acute cytotoxicity.

In experiments performed with Hololena curta spider venom, completeirreversible suppression of transmission was accomplished withoutaffecting axonal conduction presynaptically. In repeated experiments,the postsynaptic field potential failed to recover even after more thanfive hours of washing in venom-free avian tyrode solution. As mentionedabove, the active component of this venom appears to reside in afraction of about 5,000 to 7,000 daltons and appears capable of actingat even lower concentrations than the Argiope venoms discussed above.

The effect of HOI venom on the survival of cultured chick ciliaryganglion cells and their ability to extend neurites has also beenassessed. At a concentration that completely suppresses synaptictransmission in the chick cochlear nucleus, the venom did not produceincreased mortality of the cultured ciliary ganglion cells or interferewith the extension of neurites. Further, action potentials could stillbe evoked from cochlear nucleus neurons by direct electrical stimulationafter several hours of transmission blockade. These experiments indicatethat HOI venom is not generally neurotoxic at the concentrations testedand that the apparent irreversibility of transmission blockade does notresult from cochlear nucleus neurons being rendered electricallyinexcitable.

E. Comparison with other Drugs 1. Excitatory amino acid receptorblockers

Tests have been run on a large variety of excitatory amino acid receptorantagonists and agonists for their ability to suppress synaptictransmission in the chick cochlear nucleus The most potent conventionalantagonists (e.g., cis-2,3-piperidine dicarboxylic acid or-D-glutamylglycine) require concentrations in the millimolar range tocompletely suppress transmission. The agonist quisqualate alsocompletely suppresses transmission only at millimolar concentrations,presumably by depolarizing the postsynaptic neurons.

In contrast, the toxin derived from Hololena curta (HOI) completelyblocks transmission at a concentration of ten micromolar or less, makingit at least one hundred times more potent than conventional excitatoryamino acid receptor antagonists.

The actions of both AG1 and HO1 are much more persistent than those ofcurrently available excitatory amino acid (EAA) receptor antagonists,which are readily reversible. In tests using slices from chick brainincluding the cochlear nucleus, transmission blocked by application ofknown antagonists recovered completely after only 15-30 minutes ofwashout with drug-free solution.

Tests performed with these spider toxins conducted under identicalcircumstances indicate that blockade produced by AG1 persists for 45-90minutes, or three times longer. The persistence of HO1 is even morestriking in that responses show absolutely no sign of recovery evenafter several hours of washout, indicating that this compound bindsextremely tightly to the EAA receptor.

The long duration of action and the relatively tight binding areparticularly important for a basic research application involvingphysical isolation and subsequent biochemical study of EAA receptors.The toxins could be used essentially as a hook with which to grab ontothe receptor and separate it from other cellular elements, in much thesame fashion as a-bungarotoxin has been used in the isolation of theacetylcholine receptor.

This type of isolation has not proved feasible with currently availablecompounds in large part because they simply do not bind tightly enoughto serve as effective "hooks." Severe limits are placed on theunderstanding of EAA receptors, their mechanisms of function, propertiesof various subclasses, and molecular biology, by the present inabilityto isolate and directly study the receptor molecules themselves.

2. Calcium channel blockers

Receptor and voltage-activated calcium channels are of fundamentalimportance in the survival and function of virtually all cell types.Entry of calcium through such channels regulates a variety of cellularactivities including contraction of cardiovascular muscle and therelease of neurotransmitters from nerve cells. There are presently threemajor classes of organic calcium channel blockers, as opposed toinorganic blockers such as manganese or lanthanum. These organic calciumchannel blockers include: phenylalkylamines such as verapamil;benzothiazepines such as diltiazem; and dihydropyridines such asnifedipine.

The currently available organic calcium channel blockers have pronouncedactions on heart and vascular smooth muscle, although relativeselectivity for these two types of tissues varies among these compounds.A second notable features of these agents is that, although they willbind to brain tissue, they have either no effect or a relatively minoreffect on the function of neurons in the central nervous system,particularly as compared to their striking effects on heart and vascularsmooth muscle.

The two toxins derived from Agelenopsis aperta venom, AG1 and AG2, haveproperties that very clearly distinguish them from the currentlyavailable calcium channel blockers. AG1 acts primarily, if notexclusively, on neuronal calcium channels as opposed to heart orvascular smooth muscle calcium channels. This tissue selectivity isopposite to that seen in the compounds mentioned above. Furthermore, itseffects are essentially irreversible while those of currently availablecalcium channel blockers, with the exception of omega-toxin from themarine snail Conus geographus, are all reversible.

The second calcium channel blocker from Agelenopsis aperta venom, AG2,is also distinguished by its tissue selectivity or, more accurately, itsrelative lack of selectivity. This toxin is a very effective blocker ofcalcium channels in heart and, in addition, exerts an apparently equallysignificant action on neurons of the central nervous system. The actionsof AG2, unlike those of AG1, are readily reversible.

Because of the importance of calcium and calcium channels to thefunction of various cell types, there are a variety of potentialtherapeutic applications of compounds within the scope of the presentinvention. Calcium influx through channels mediates contraction of heartand vascular muscle. Calcium channel blockers, therefore, tend to relaxboth heart and vascular muscle and damp arrhythmic cardiac activity.

Accordingly, calcium channel blockers are presently used in treatment ofseveral cardiovascular disorders including angina, arrhythmia,hypertension, and cardiomyopathy. In addition, calcium antagonists tendto inhibit platelet aggregation and so may have application in coronaryocclusion and coronary vasospasm. Calcium channel blockers have alsobeen shown to exert an antiatherosclerotic action.

Analyses performed on AG2 indicate that it is a very effective blockerof calcium channels in heart muscle and, furthermore, that it exertsthis effect with few if any additional actions. This is in clearcontrast to currently available compounds which, in addition to blockingcalcium channels, typically have other actions such as increasing ordecreasing the flow of potassium through its channels. This specificityof the effects of AG2 suggests that it would have the desiredtherapeutic action on calcium channels with fewer unwanted side-effectsarising from the use of currently available compounds.

Insofar as AG2 has a significant effect on calcium channels in thecentral nervous system, it has several applications to neurologicaldisorders. There is mounting evidence that epileptic activity in thebrain and resulting damage to neurons may involve calcium currentsflowing through receptor or voltage-activated calcium channels. As aresult, calcium channel blockers are expected to be effective inblocking seizures and preventing neuronal damage associated withseizures. Such a seizure effect is clearly indicated by the experimentsdescribed above using rats and AG2. Damage to neurons resulting fromstroke seems to involve the excessive accumulation of calcium withinneurons following hypoxia and the cytotoxic effects of calcium.

III. EXAMPLES

The following examples are given to illustrate particular compositionsand methods within the scope of the present invention but they are notintended to limit the scope of the present invention.

EXAMPLE 1

A spider toxin within the scope of the present invention was isolatedfrom the Agelenopsis aperta spider. Spider venom was obtained from, andspecies identification provided by, Spider Pharm, Inc. of Black CanyonCity, Ariz. Agelenopsis aperta spiders were electrically milked using amethod that employs safeguards to prevent contamination of venom byabdominal regurgitate or hemolymph. Venom was diluted 1 to 10 with avianTyrode solution (140 mM NaCl, 4 mM KCl, 4 mM NaHCO₃, 1 mM MgSO₄, 3 mMCaCl, 1.2 mM NaH₂ PO₄, 10 mM HEPES, 10 mM glucose) and fractionated bygel filtration using Bio-Gel P-10 and a 0.7 ×30 cm column and collectedin 0.5 ml fractions. These fractions were assayed for blockade ofsynaptic transmission using the electrophysiological methods describedbelow.

HPLC separation of gel filtration fractions was performed using a VydacC-18 reverse phase column. Components were eluted from the column over aperiod of 60 minutes using a 0-60% linear gradient of 60% acetonitrilein 0.1% trifluoroacetic acid. Elution was monitored by absorbancedetection at 214 nm. Peaks were collected manually, dried down, storedat -20° C., and then reconstituted by varying concentrations with aviantyrode before use.

For gel electrophoresis, a 15 uL sample of each gel filtration fractionwas mixed with 7.5 ul of 3× sample buffer (18% 1M tris-HCL, pH 6.8, 15%2 mercaptoethanol, 30% glycerol, 7% sodium dodecyl-sulfate, 0.001%bromphenol blue) and the entire sample was loaded onto a 10% to 20%gradient polyacrylamide gel using the slab method. Electrophoresis wasperformed at 20 watts constant power for three hours. Gels were stainedwith Coomassie blue.

The toxin so isolated had a molecular weight of approximately 6,000 asestimated from SDS polyacrylamide gels. The toxin was bath-applied tocochlear nucleus neurons in an in vitro preparation of chick brain stem.Upon stimulation of the cochlear nerve innervating the cochlear nucleus,it was found that the toxin blocked transmission between the cochlearnerve afferents and the cochlear nucleus neurons.

After washout of the toxin with toxin-free Tyrode solution, blockadepersisted for the useful life of the preparation (about 8 hours). Evenextremely low concentrations of about 0.1 uM produced complete blockadeif applied for extended periods of about one hour. The toxin alsoappeared to be quite potent in that exposure of the tissue to a 0.8 uMsolution for even two minutes resulted in complete blockage oftransmission.

The results suggest that blockage using the toxin so obtained isirreversible, or at the very least the toxin binds unusually tightly toits targets.

EXAMPLE 2

The high molecular weight Agelenopsis aperta toxin described in Example1 was obtained using the same procedure as described in Example 1.Gel-filtration fractions having the effects described in Example 1 werediluted 1:150 with Tyrode and bath-applied for one minute to an in vitropreparation of the chick brain stem. This brief exposure to dilute toxinproduced partial (about 50%) blockade of transmission.

These partial blocks were stable for at least one hour and wereunaffected by increases or decreases in the rate of cochlear nervestimulation over a range of 0-30 Hz. Partial blockade, however, waslargely reversed by increasing extracellular calcium from 3 to 9 mM.Subsequent reduction of extracellular calcium back to 3 mM caused thepostsynaptic response to revert to its previous level of partialblockade.

The result so obtained indicates that this toxin acts presynaptically toproduce long-lasting blockade of transmission. The finding that theeffects of this toxin are independent of stimulation frequency suggeststhat the toxin does not act primarily on synthesis or reuptake oftransmitter The inverse relationship between extracellular calciumconcentration and the blocking effects of the toxin, indicates that thetoxin likely acts on calcium channels. This action could be exerted onpresynaptic calcium channels necessary for the release of transmitterand/or on postsynaptic calcium channels involved in the response of thecochlear nucleus neurons to synaptic stimulation.

EXAMPLE 3

The toxin AG1 described in Examples 1 and 2 is obtained in the mannerdescribed above from Agelenopsis aperta spider venom.

The toxin so obtained is applied and a complete blockade of synaptictransmission is achieved as described in Example 1. EAA agonistsquisqualic acid and kainic acid are then individually bath-applied tothe cochlear nucleus neurons at concentrations of 5 mM and 50 uM,respectively.

Application of these agonists at such concentrations normally reducesthe ability of cochlear nucleus neurons to respond to direct electricalstimulation, presumably by depolarizing them by their action on EAAreceptors. When applied in the presence of AG1 toxin the same effect isseen; that is, after 10 minutes of application of either quisqualic ofkainic acids in the presence of AG1 toxin the response of cochlearnucleus neurons to direct antidromic stimulation is reduced by about75%. The degree and time-course of this effect are not significantlydifferent from those observed when either quisqualic of kainic acid isapplied in the absence of AG1 toxin.

The result so obtained indicates that this toxin does not exert itsblocking effects on synaptic transmission by a direct action on EAAreceptors on the postsynaptic neuron but rather by a direct action ofthe toxin on calcium channels as suggested in Examples 1 and 2. If itwere acting directly on EAA receptors it would be expected that thetoxin would also block the effects of directly applied EAA agonists,such as quisqualic or kainic acid. The obtained result runs counter tothis expectation.

EXAMPLE 4

A spider toxin within the scope of the invention was isolated from theAgelenopsis aperta spider. Spider venom was obtained from Spider Pharm,Inc. of Black Canyon City, Ariz. Agelenopsis aperta spiders wereelectrically milked using a method that employs safeguards to preventcontamination of venom by abdominal regurgitate or hemolymph. Venom wasdiluted 1 to 10 with avian tyrode solution and fractionated by gelfiltration using BioGel P-10 and a 0.7×30 centimeter column andcollected in 0.5 mil fractions.

HPLC separation of gel filtration fractions was performed using a VydacC-18 reverse phase column. Components were eluted from the column over aperiod of 60 minutes using a 0-60% linear gradient of 60% acetonitrilein 0.1% trifluoroacetic acid. Elution was monitored by absorbancedetection at 214 nm. Peaks were collected manually, dried down, storedat -20° C., and then reconstituted by varying concentrations with aviantyrode before use.

For gel electrophoresis, a 15 ul sample of each gel filtration fractionwas mixed with 7.5 ul of 3× sample buffer (18% 1M tris-HCL, pH 6.8, 15%2 mercaptoethanol, 30% glycerol, 7% sodium dodecyl-sulfate, 0.001%bromphenol blue) and the entire sample was loaded onto a 10% to 20%gradient polyacrylamide gel using the slab method. Electrophoresis wasperformed at 20 watts constant power for three hours. Gels were stainedwith Coomassie blue.

The toxin so isolated had a molecular weight of approximately 600 asestimated from SDS polyacrylamide gels.

Cultured single heart cells were prepared from 9 to 10 s day oldembryonic chick hearts (ventricles) by standard techniques. The cellswere dispersed in sterile HMEM (Hanks Minimum Essential Medium, Gibco)containing 0.1% trypsin and 1.8 nm Ca²⁺. The cell digest was collectedthrough sterile gauze, pooled, and centrifuged at 170 g for 10 minutes.Single cells were resuspended in culture medium and centrifuged again inorder to wash out the trypsin.

Cells were then placed in a plastic dish for 30 minutes at 37° C. toallow the fibroplasts to attach themselves to the plastic dish. In orderto eliminate the fibroplasts, the heart cells remaining in suspensionwere transferred to a new plastic dish leaving the attached fibroplasts.The culture medium was made of HMEM containing 5% fetal bovine serum(Gibco) and 50 IU/ml penicillin-G-potassium (Ayerst). The cultured heatcells were kept in a 5% CO₂, 95% O₂, incubator at 37° C. Cultured singleheart cells were used after 1 to 2 days for whole-cell voltage clamprecordings.

In order to perform whole-cell voltage clamp recording, patch pipetswere prepared by pulling capillary tubes in two steps using a verticalpuller. The pipets were filled with a solution containing, in millimolesper liter: NaCl 20: KCl 130;MgCl₂ 2; EGTA 5; HEPES buffer 5; and glucose5 (pH 7.4) for recording action potentials in with solutions containing:K-aspartate, 130; TEA, 20;MgCl₂, 2; EGTA, 5; ATP, 0.3; cAMP, 0.03; HEPESbuffer, 5, and glucose, 5 (pH 7.4), for the study of ISI usingwhole-cell voltage clamp technique. The cells were superfused withextracellular solution containing (in millimoles per liter) forrecording action potentials; NaCl, 130; KCl, 5.4; CaCl₂, 2.2; MgCl₂,0.2; HEPES buffer, 5, and glucose, 5 (pH 7.4) at 22° C., and for thestudy of I_(CA) ; TEA, 130; 4-aminopyridine, 5.4; CaCl₂, 1.8;MgCl, 1.03;HEPES buffer, 5, and glucose, 5 (pH 7.4) at 35° C. The pipet resistancesranged from 2 to 50 megaohms, and the seal resistance ranged from 20 to10 G ohms.

A patch clamp amplifier was used for the voltage clamp experiments.

It was found that two of the gel-filtration fractions containinglow-molecular weight had significant acute effects on the ventricularcell action potentials. One of these two fractions was abou twice aspotent as the other in producing the effect described as follows. Theresting membrane potential was -80 mV and superfusion with Tyrodesolution containing a 1 to 10 dilution of the gel filtration fractioncontaining the toxin progressively decreased the action potentialduration and overshoot. This effect indicates blockade of the slowinward calcium current (I_(si)). There was no effect on resting membranepotential or +Vmax, indicating that the toxin did not affect the inwardfast sodium current.

EXAMPLE 5

In this example, the procedure outlined in Example 4 was followed.

In order to more directly assess the effect of the toxin on the calciumcurrent, whole-cell voltage clamp was used and I_(si) was recorded inthe absence of Na and K ions. The most potent of the gel-filtrationfractions, as described in Example 4, was applied to chick heart cellsat various concentrations. A dilution of 1:150 decreased I_(si) within 4minutes and a steady state was established after 7 minutes. The effectwas fully reversible upon washout. A dilution of 1:100 further decreasedI_(si) and that current was completely blocked after 10 minutes ofexposure to a 1:10 dilution.

These results indicate a specific, reversible blockade of the slowinward calcium current in heart muscle by the AG2 toxin.

EXAMPLE 6

In this example, the procedure outlined in Example 4 was followed. Themost potent gel-filtration fraction having the effects on heartdescribed in Examples 4 was further purified using high performanceliquid chromatography (HPLC) according to the procedure described inExample 4. The HPLC profile revealed seven major peaks. Each of thesewere collected and assayed for effects on the I_(si) using voltage-clampas described in Example 4. Only one of the HPLC peaks or fractions hadany significant effect. At a 1:10 dilution, that fraction progressivelydecreased I_(si) within 7 minutes and completely blocked it within 9minutes. This effect was fully reversible upon washout of the toxin.

These results indicate that the toxin molecule producing the observedeffect on calcium currents in heart has been highly purified from theoriginal whole Agelenopsis aperta venom.

EXAMPLE 7

Low molecular weight toxin (AG2) is isolated as described in theprocedure outlined in Example 4. Toxin so isolated is used to treathuman patients suffering from angina pectoris of any of three types:stable angina on effort, unstable angina at rest, and variant angina dueto vasospasm. Treatment with AG2 will decrease the frequency of anginalattacks and produce prolonged depression of the ST segment of theelectrocardiogram which can significantly prolong exercise duration.During exercise, angina is diminished in severity or abolished and therequirement for nitroglycerine therapy is diminished or abolished. AG2will produce these beneficial cardiovascular effects by one or more ofthe following actions: decreased systemic vascular resistance andarterial blood pressure; a direct negative inotropic effect, peripheralvasodilation, and a direct negative inotropic effect, peripheralvasodilation, and a direct negative chronotropic effect, all of whichdecrease myocardial oxygen consumption; dilation of coronary arteries,which improves myocardial blood supply; a specific effect on myocardialmetabolism; conduction delay interfering with the reentrant cycle oftachycardia.

EXAMPLE 8

Toxin AG2 is isolated as described in Example 4 and administered tohuman patients suffering from hypertropic cardiomyopathy. Markedsymptomatic relief is noted and one or more of the following specificimprovements in cardiac function will be observed: the abnormal leftventricular diastolic properties are reverted; the prolonged leftventricular isovolumic relaxation time will decrease, left ventricularpressure decay will be accelerated, and the depressed left ventricularfilling increases significantly. Left ventricular thinning is alsoimproved and there is a significant decrease in left ventricularend-diastolic pressure and a downward shift in the left ventriculardiastolic pressure-dimension curve without any effect on systolicfunction.

EXAMPLE 9

Toxin AG2 is isolated as described in Example 4 and administered tohuman patients suffering from systemic hypertension. Regular treatmentwith AG2 is found to provide symptomatic relief and effective control ofthis disorder by significantly decreasing arterial blood pressurethrough one or more of the following mechanisms: decreased peripheralarterial resistance, decreased pulmonary arterial resistance, anddecreased pressor and aldosterone response to angiotensin II.

EXAMPLE 10

Toxin AG2 is isolated as described in Example 4 and administered, incombination with other drugs, to human patients suffering fromcongestive heart failure. Treatment with AG2 produces symptomatic reliefand a significant improvement in cardiac function in these patients, asindicated by one or more of the following indices: a decrease in meanpulmonary artery, mean aortic, and left ventricular end-diastolic bloodpressures, decreases in systemic and pulmonary vascular resistance, andan increase in cardiac index; increases in left ventricular ejectionfraction and stroke volume; decreases in left ventricular volumes; areduction in myocardial oxygen requirements; improvements in diastolicperformance.

EXAMPLE 11

Toxin AG2 is isolated as described in Example 4 and administered tohuman patients suffering from pulmonary hypertension. Significantclinical improvement is obtained, as measured by relief of symptions andone or more of the following indices of cardiovascular function:decreased pulmonary vascular resistance; increases in cardiac output;and increases in right ventricular ejection fraction with decreases inright ventricular volumes.

EXAMPLE 12

AG2 is isolated as described in Example 4 and administered to humanpatients suffering from peripheral vascular disease (e.g., Raynaud'sDisease). The frequency of disease attacks is reduced, as is theseverity of the attacks when they occur.

EXAMPLE 13

AG2 is isolated as described in Example 4 and administered to humanpatients undergoing transluminal angioplastic surgery. By its transientregional cardioplegic effects and its production of increased coronaryblood flow, AG2 preserves the ventricular myocardium from ischemiainduced by the surgical procedure and thereby prolongs inflationperiods, an important determinant of the success of transluminalangioplasty.

EXAMPLE 14

AG2 is isolated as described in Example 4 and administered to humanpatients with symptomatic atherosclerosis or patients at elevated riskof developing atherosclerosis. AG2 will decrease existingatherosclerotic stenosis of blood vessels (with ensuing symptomaticrelief) and prevent or reduce development of atherosclerosis by one ormore of the following actions: prevention of calcium accumulation inarterial walls, reduction in the force of contractility and endothelialdamage caused by the bloodstream, decreased blood pressure, andanti-platelet aggregating effects.

EXAMPLE 15

AG2 is isolated as described in Example 4 and administered to patientssuffering from cardiac arrhythmia of any of the following types: sinustachycardia, supraventricular tachycardia, atrial fibrillation andflutter, ventricular premature contractions and ventricular tachycardia,premature atrial contractions, and chronic atrial fibrillation.Treatment with AG2 will provide symptomatic relief through one or moreof the following actions slowing of the sinus rate, slowing of thereentrant cycle, suppression of extrasystoles responsible for thearrhythmia, a parasympathomimetic effect, or induction of ventricularectopy interfering with the tachycardia cycle, decreasing the amplitudeof action potentials in the upper and middle zone of theatrioventricular node and lengthening the effective refractory period ofthe atrioventricular node, which lead to slowing of the atrioventricularconduction time; interruption of a tachycardia reentry mechanisminvolving a slow response action potential qr a depressed fast responseaction potential or a slow-channel dependent triggered automaticity.

EXAMPLE 16

AG2 is isolated as described in Experiment 4 and administered to humanpatients suffering from achute myocardial ischemia or infarction.Significant symptomatic relief and protection of the myocardium fromcellular damage is observed. These beneficial actions are due to AG2'svasodilation effects, negative inotropic effect and/or inhibition ofcalcium accumulation in the myocardium upon reperfusion.

EXAMPLE 17

AG2 toxin was isolated according to the procedure outlined in Example 4.Rats are intravenously injected with 12 mg/kg dose of the convulsantkainic acid. This is followed after five minutes with an I.V. injectionof AG2 toxin in an amount equivalent to that derived from 50 ul of wholevenom by the procedure outlined in Example 4. Control subjects receiveonly the kainic acid injection. Control subjects begin to show seizureactivity after 10-15 minutes. By 30 minutes they show frequent (aboutone every two minutes) clonic-tonic seizures and after one hour showvirtually continuous tonic-clonic seizures (status epilepticus). Bythree hours about 50% of control subjects have died. About 80% ofcontrol subjects die within 36 hours.

Subjects receiving the same dose of kainic acid but a subsequentinjection of AG2 as described above, however, show virtually no seizurebehavior until about 90 minutes after the kainic acid injection. Seizurebehavior then increases over a period of about one hour, but only about15% of these animals ever enter status epilepticus and none die evenafter 48 hours.

These results show that AG2 effectively counters in the convulsantactivity of kainic acid. The gradual onset of a lower level of seizureactivity after about 90 minutes is commensurate with the reversibleaction of AG2 observed in brain and heart.

Kainic acid induced seizures have been used as a model for humantemporal lobe epilepsy. In addition, abnormal calcium-mediatedelectrical activity in neurons has been implicated in several types ofepilepsy. Therefore, these results indicate that AG2 or its analoguesmay prove effective in the clinical control of human epilepsy.

EXAMPLE 18

Venom was obtained and isolated from the Argiope aurantia spider. Thevenom used was obtained from Spider Pharm of Black Canyon City, Ariz.The venom was electrically milked from the spiders using a method whichemploys safeguards to prevent contamination of the venom by abdominalregurgitate and hemolymph.

The first stage in separating the venom was carried out using gelfiltration chromatography. Venom was diluted 1 to 10 with phosphatebuffered saline and passed over BioGel P-6 using a 0.7×10 cm columneluted with the same buffer Fractions were collected in 1.0 ml aliquotsand stored at -20° C.

The protein content of both whole venom and P-6 fractions was determinedusing conventional methods and measuring absorbance with a GilfordG-2600 spectrophotometer.

Ten microliters of venom that had been diluted 1 to 10 with phosphatebuffered saline was mixed with 5 ul of 3× sample buffer. Afterincubation at 37° C. for 15 minutes 5 ul of each sample was applied to a10% to 20% gradient polyacrylamide gel using the slab gel method knownin the art and the discontinuous buffer system and electrophoresed at 20watts constant power for 3 hours.

Gels were stained with Coomassie blue staining solution and destained in10% methanol/10% acetic acid solution. The same procedure was used forelectrophoresis of venom fractions except that a 15 ul sample of eachfraction was mixed with 7.5 ul of 3× sample buffer and the entire samplewas loaded onto the gel. Molecular weights of various fractions wereestimated from known standards run on the same gel.

High performance liquid chromatography was performed on the lowmolecular weight component of the venom using a Vydac C-18 reverse phasecolumn. Components were eluted from the column over a period of 60minutes using 0% to 60% linear gradient of 60% acetonitrile in 0.1%trifluoroacetic acid. Elution was monitored by absorbance detection at214 nm. Peaks were collected manually, dried down, and stored at -20° C.

In order to test the effects of the spider venom, late embryos orhatchling chickens were used in all experiments Brain stems were removedand maintained in vitro using methods known and described in the art,except that the temperature in the recording chamber was held at 32° C.The tissue was superfused in avian Tyrode solution at about 1 ml perminute.

The field potential evoked by direct electrical stimulation of thecochlear nerve was recorded with a 4M potassium acetate electrode. Theshort latency component of the field potential reflects the compoundaction currents of cochlear nerve axons. This afferent volley isfollowed by a negativity representing the postsynaptic response of NMneurons.

It was found that Argiope aurantia venom blocked synaptic transmissionbetween cochlear nerve axons and their target neurons in the cochlearnucleus (nucleus magnocellularis) without affecting the presynapticvolley. When applied in avian Tyrode solution at a 1 to 300 dilution anda superfusion rate of about 1 ml per minute, whole venom completelyblocked the postsynaptic component of the field potential within 15minutes. Recovery of responses was variable, requiring from 45 minutesto 2 hours. Total blockade with a similar onset and recovery time wasseen in a single test of venom diluted 1:600.

Electrophysiological assays of gel-filtration fractions revealed a veryclear concentration of activity in the lowest molecular weight fraction.At concentrations equivalent to the 1:300 and 1:600 dilutions of wholevenom tested as described above, this gel-filtration fraction causedcomplete blockade of postsynaptic responses within 15 minutes. Recoveryof responses upon washout of this partially-purified toxin occurred fromabout 45 minutes to 2 hours, as with the whole venom.

Components of this active gel-filtration fraction were further separatedusing HPLC. Three major peaks and a number of smaller components wereseen in the HPLC profile. Electrophysiological assays of the peaksindicated that the largest was the most active. Material in that peaksuppressed the postsynaptic response by almost 80% within 5 minutes. Thesecond largest peak also showed some activity, although that componentproduced only a 40% block. No other components had any significanteffect on transmission.

As judged by their migration in Bio-Gel P6, and on SDS-PAGE, the toxiccomponents are of relatively low molecular weight. Material from peaks Band C was examined by fast atom bombardment mass spectrometry. Peak Bwas a mixture with the two principal components having strong molecularions (MH⁺) at m/z 637 and 646. Peak C was also a mixture, the maincomponent giving a strong molecular ion (MH⁺) at m/z 660 In all casesthere were significant peaks corresponding to the doubly-protonatedmolecular ions at m/z=(MH₂ ⁺⁺)/2, as indicated by half-mass unitspacings.

The ultraviolet absorption spectrum of peak C showed maxima in the nearu.v. at 267, 282, and 292 nm, suggesting the presence of a modifiedaromatic residue. Amino acid analysis after hydrolysis by 6N HCl for 20hours at 105 C gave Arg and Asp in a ratio of approximately 2:1, plus anunknown compound. High-voltage electrophoresis at pH 6.5 stainedpositively with ninhydrin and Pauli reagent (specific for His andphenols such as Tyr), but gave no reaction with the Ehrlich stain forTrp. Its mobility relative to peptides of known mass and chargeindicated that it carries a net charge of +3 at this pH. Sequentialdegradation for several cycles in a protein sequencer gave PTH-Arg inthe first cycle, but no recognizable derivatives in subsequent cycles.

It was further found that there is no evidence that blockade oftransmission in the chick cochlear nucleus is dependent on stimulationfrequency. Increasing the rate of cochlear nerve stimulation from 0.2 to10 Hz during application of toxin caused no obvious increase in the rateof blockade.

In summary, Argiope Aurantia toxin was isolated and found to produce areadily reversible blockade of transmission in chick cochlear nucleus.

EXAMPLE 19

Toxin of the Hololena curta spider was obtained and purified using theprocedure described in Example 4. Using the electrophysiologicaltechniques of Example 16, the toxin so obtained was applied to the chickcochlear nucleus. Assays of all venom fractions obtained bygel-filtration revealed that certain adjacent fractions suppressedsynaptic transmission in the chick cochlear nucleus.

The most potent of the gel-filtration fractions, when applied at adilution of 1:15, completely blocked postsynaptic responses within 10minutes. Even dilutions of 1:150, if applied to the tissue for longerperiods (about 30 minutes) caused a complete blockade of transmission.At no time was there any evidence of recovery of responses for theuseful life of the preparation (about 8 hours). The molecular weight ofmaterial in the active gel-filtration fractions was 5,000-10,000 daltonsas estimated by comparison with known standards run on the same SDSpolyacrylamide gels.

These results suggest that this toxin from the Hoblena curta spidervenom is a patent blocker of transmission in the chick cochlear nucleusmediated by EAA receptors. The results also indicate that the action ofthe toxin is irreversible or at least very unusually long lasting.

EXAMPLE 20

The high molecular weight toxin described in Example 19 is obtained inthe same manner from Hololena curta spider venom.

The toxin so obtained is used as a ligand in affinity chromatography toisolate and purify EAA receptors or receptor-channel complexes; thefollowing general procedures are used. Activated polyamide is used as asorbent-carrier and HO I is used as a ligand. One gram of activatedpolyamide powder treated by glutaraldehyde is incubated for 24 hourswith 11 mg of the toxin in a buffer. The remaining free carrier aldehydegroups are blocked by ethanolamine. Crude synaptosomal preparations fromhuman or animal brain are sonicated. The sonicated membrane fractionsare incubated with this affinity sorbent in the presence of NaNO₂ for 48hours. The unbound molecules are carefully washed out and then a pHgradient is used to elute specific molecules bound to the toxin. Thesemolecules are further purified by conventional techniques and theiridentity as EAA receptors or receptor-channel complexes confirmed byinsertion of the molecules into artificial membranes with subsequentpharamcophysiological testing.

EXAMPLE 21

The high molecular weight toxin described in Examples 1-3 (AG1) isobtained in the manner described above from Agelenopsis aperta spidervenom.

The toxin so obtained is applied for the purpose of labeling calciumchannels or receptors in neurons or other cell types. A radioactivelabel (such as ³ H, ¹⁴ C, or ¹²⁵ I) is incorporated in the toxinmolecule. Binding of the labeled toxin is then assayed usingautoradiography of tissue sections or quantifications (usingscintillation counting) of binding to various tissue extracts such assynaptosomal or membrane preparations. Autoradiography of brain tissuesections labeled with the radioactive toxin reveals the regionaldistribution of the calcium channels or receptors to which the toxinbinds. (A similar result is obtained by observing the binding pattern ofthe toxin conjugated to a fluorescent label.) The binding of the toxinsto tissue extracts under various conditions, such as the presence ofother drugs, provides information regarding the pharmacology of thecalcium channel or receptor to which the toxin binds.

EXAMPLE 22

The toxin obtained in the manner described in Example 16 is applied anda complete blockade of synaptic transmission is achieved as described inExample 6. EAA agonists quisqualic and kainic acid are then individuallyboth applied to the cochlear nucleus neurons at concentrations of 5 mMand 50 mM, respectively.

Applications of these agonists at such concentrations normally reducesthe ability of cochlear nucleus neurons to respond to direct electricalstimulation, presumably by displaying them by their action on EAAreceptors. When applied in the presence of Argiope aurantia toxin,however, these agonists have no significant effect; that is, even after20 minutes of application, these agonists have no effect on the responseof cochlear nucleus neurons to direct antidromic stimulation.

The result so obtained indicates that this toxin exerts its effect byacting as an excitatory amino acid receptor antagonist.

EXAMPLE 23

The toxin obtained in the manner described in Example 19 is applied anda complete blockade of synaptic transmission is achieved as described inExample 6. EAA agonists quisqualic and kainic acid are then individuallyboth applied to the cochlear nucleus neurons at concentrations of 5 mMand 50 mM, respectively.

Applications of these agonists at such concentrations normally reducesthe ability of cochlear nucleus neurons to respond to direct electricalstimulation, presumably by displaying them by their action on EAAreceptors. When applied in the presence of Hololena curta toxin,however, these agonists have no significant effect; that is, even after20 minutes of application, these agonists have no effect on the responseof cochlear nucleus neurons to direct antidromic stimulation.

The result so obtained indicates that this toxin exerts its effect byacting as an excitatory amino acid receptor antagonist.

EXAMPLE 24

HO1 isolated as described in Example 19 is used to treat humaninfections with helminthic worms of the genus Schistosoma. In thisexample, infection with S. japohicum is considered. Treatment with HO1during acute prmary infection results in clinical improvement of one ormore of the following symptoms through reduction in the patient'sparasiate load: high spiking fever, chills, cough, urticaria,generalized lymphadenopathy, tender hepatosplenomegaly, eosinophilicleucocytosis, intestinal wall ulcers, abdominal pain, and bloody stools.Treatment with HO1 during later stages of infection reduces one or moreof the following symptoms by reduction of the patient's larval load:engorgement of superficial abdominal venous ascites, splenomegaly,anemia, leukopenia, and thrombocytopenia.

IV. SUMMARY

In summary, it can be seen that the methods and compositions of theabove invention accomplish the objectives set forth above. Inparticular, the present invention provides channel blockers orexcitatory amino acid receptor blockers which are selectively reversibleor irreversible in their effects and which can be used as research toolsor in a clinical setting. In particular, the spider toxins of thepresent invention can be used as channel blockers or excitatory aminoacid receptor blockers in the central nervous system, the cardiovascularsystem, or both systems. In addition, either reversible or irreversibleeffects can be obtained using the present invention.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

What is claimed and desired to be secured by United States LettersPatent is:
 1. A composition functioning as an excitatory amino acidreceptor blocker comprising at least one toxin extracted from Argiopeaurantia spider venom, said at least one toxin being capable offunctioning as an excitatory amino acid receptor blocker.
 2. Acomposition as defined in claim 1 wherein the effects of the toxin arereversible.
 3. A composition as defined in claim 1 wherein the effectsof the toxin are irreversible.
 4. A composition as defined in claim 1wherein the toxin blocks excitatory amino acid receptors in the nervoussystem.
 5. A composition as defined in claim 1 wherein the toxin has amolecular weight of from about 500 to about 1,000 daltons.
 6. Acomposition as defined in claim 5 wherein the effects of said toxin arereversible.
 7. A composition functioning as an excitatory amino acidreceptor blocker comprising a toxin extracted from the venom of theArgiope aurantia spider and having a molecular weight of from about 500to about 1,000 daltons.
 8. A composition as defined in claim 7 whereinthe toxin has a molecular weight of from about 650 to about 670 daltons.9. A composition as defined in claim 8 which produces a readilyreversible blockage of transmission in chick cochlear nucleus.
 10. Aninsecticide consisting essentially of at least one toxin extracted fromthe whole venom of the Agelenopsis aperta spider.
 11. An insecticide asdefined in claim 10 wherein the toxin has a molecular weight of fromabout 500 to about 1,500 daltons.
 12. An insecticide as defined in claim10 wherein the toxin has a molecular weight of from about 5,000 to about10,000 daltons.
 13. An insecticide comprising a toxin extracted from thewhole venom of the Hololena curta spider and having a molecular weightof from about 5,000 to about 7,000 daltons.
 14. An insecticidefunctioning as an excitatory amino acid receptor blocker comprising atoxin extracted from the Argiope aurantia spider and having a molecularweight of from about 500 to about 1,000 daltons.
 15. An anthelminticfunctioning as an excitatory amino acid receptor blocker comprising atoxin extracted form the Argiope aurantia spider and having a molecularweight of from about 600 to about 700 daltons.
 16. A composition asdefined in claim 1 wherein said toxin has a molecular weight ofapproximately 660 daltons.
 17. A composition as defined in claim 16wherein the effects of said toxin are reversible.
 18. A method ofmanufacturing an excitatory amino acid receptor blocker comprising thesteps of:(a) obtaining at least one Argiope aurantia spider; (b)obtaining venom from said at least one spider; (c) separating the toxinsof the venom so obtained; (d) recovering an individual toxin separatedfrom the venom; and (e) combining said isolated toxin with apharmaceutically acceptable carrier such that the toxin is capable offunctioning as an excitatory amino acid receptor blocker.
 19. A methodas defined in claim 18 wherein the toxin recovered has a molecularweight of from about 500 to about 1,000 daltons.
 20. A method ofadministering an excitatory amino acid receptor blocker comprising thesteps of:(a) obtaining an excitatory amino acid receptor blockercomposition comprising at least one toxin isolated from the venom of theArgiope aurantia spider, said isolated toxin being combined with apharmaceutically acceptable carrier such that the composition is capableof functioning as an excitatory amino acid receptor blocker; and (b)administering said excitatory amino acid receptor blocker composition toa human patient.
 21. A method of administering an excitatory amino acidreceptor blocker as defined in claim 20 wherein the at least one toxinisolated from the venom of the Argiope aurantia spider has a molecularweight of from about 500 to about 1,000 daltons.